The SARS outbreak of 2002/2003 demonstrated the lethality and communicability of emerging infectious agents. Clinical presentation of SARS is similar to other respiratory infections, complicating diagnosis, however diagnosis by molecular means differentiates SARS from less dangerous respiratory infections. Under contract to the US Department of Defense, Idaho Technology developed the RAZOR, a battery-operated hand-portable PCR instrument utilizing fluorescence to detect -specific amplification products and software to automatically call samples as either positive or negative. This application proposes four specific aims involving the transforming the military RAZOR to the civilian PathFinder with an initial application to differentiate common respiratory viruses from SARS.
Specific Aim I involves engineering changes to the existing instrument to allow high-resolution thermal melting curve analysis. This includes changes to the optical system such that detection using the novel dsDNA binding dye LCGreen is possible and changes to the heaters to allow fine temperature control.
Specific Aim 2 modifies Idaho Technology's existing manual DNA purification protocol to purify RNA from human nasopharangeal aspirate samples (the matrix of choice for diagnosis of respiratory infections).
Specific Aim 3 Multiplex analysis of high-resolution thermal melting curves requires that the melting characteristic of individual amplification products be unique. A set of amplicons specific for each of the RNA respiratory viruses: RSV, influenza A and B and the SARS coronavirus are developed with each have well-separated, readily differentiated melting profiles. Sets of primers to generate these amplification products are tested in combinations necessary for multiplexing to a level of 4. Lyophilized reagents for performing the reverse transcription/PCR reactions are developed and are an integral part of platform.
Specific Aim 4 proposes to integrate PathFinder into a workstation automating nucleic acid preparation and seamlessly preparing the PCR reaction. Several possible methodologies for cell lysis, nucleic acid extraction and purification in a low power format are considered. ? ?
| Poritz, Mark A; Blaschke, Anne J; Byington, Carrie L et al. (2011) FilmArray, an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection. PLoS One 6:e26047 |
